The broad, long-term objectives of the project are to setup and optimize a high-throughput screen (HTS) for small molecule inhibitors of association between carboxy-terminal region of N-type calcium channel and Mintl-PDZ1 domain. N-type Ca2+ channels play a predominant role in the sensation of pain. Opening of presynaptic N-type (CaV2.2) Ca2+ channels results in Ca2+ influx into presynaptic terminal, synaptic vesicle fusion and secretion of a neurotransmitter. In previous biochemical experiments we discovered the association of carboxy-terminal regions of CaV2.2 subunit with Mintl-PDZ1 domain and demonstrated that association of CaV2.2 with Mintl-PDZ1 is important for synaptic targeting and synaptic function of N-type Ca2+ channels. Based on these results we proposed that small molecule inhibitors of association between CaV2.2 and Mintl-PDZ1 may be used to treat chronic pain (neuropathic pain, inflammatory pain, cancer pain). To identify such inhibitors, we intend to: Use scintillation-proximity assay (SPA, Aim 1) and homogeneous time-resolved fluorescence resonance energy transfer assay (HTRF, Aim 2) to screen for small-molecule inhibitors of association between CaV2.2carboxy-terminal fragment (NC peptide) and Mintl-PDZ1 domain expressed as GST-fusion protein (GSTMintl-PDZ1). The proposed SPA and HTRF HTS screens will be optimized with the test library of 8,000compounds to yield a Z factor of at least 0.5 (Aim 3).Assay that yields most robust results (SPA or HTRF) will be used in HTS screen with a full library of150,000-500,000 compounds. Biological activity of compounds identified in the full HTS screen will be tested in whole animal pain assays (formalin, hot plate, tail flick). [unreadable] [unreadable]